Major histocompatibility complex (MHC) molecules play a central role in determining the specific viral peptides recognized by T lymphocytes. A CD8+ cytotoxic T lymphocyte clone obtained from a SIV-infected monkey was previously shown to recognize a 9 amino acid peptide in the second hypervariable region of the SIV envelope glycoprotein. Characterization of the MHC molecule responsible for presenting this epitope will facilitate efforts to understand the role that CTL play in controlling viral replication and analysis of candidate vaccines. Class I MHC molecules from this animal have been molecularly cloned and expressed in a human B cell line deficient in class I expression, and characterized by one-dimensional isoelectric focusing. We used modified PCR primers that were derived from relatively conserved 5' and 3' untranslated flanking regions of MHC mRNA, and successfully amplified a 1.2 Kb PCR fragment from the Rhesus EBV transfected B cell line 19166. The pool of MHC class I molecules was subcloned into a eurkayotic expression vector and expressed in the C1R cell line. Isoelectric focusing of immunoprecipitated class I molecules has been carried out on transfected C1R cells to characterize cloned class I molecules. Use of this technique should facilitate more rapid characterization of class I molecules responsible for presenting CTL epitopes.